Biology subjects

Tholey, A.

Publications and source records attributed to Tholey, A..

At least 19 recordsLinked to original sources

bioRxiv2026

An effector protein that protects a fungal pathogen from the plant microbiota during host colonisation

Zymoseptoria tritici is the causal agent of Septoria tritici blotch, one of the most economically important wheat diseases worldwide. One of the few cloned wheat resistance genes against Z. tritici, Stb6, recognizes the secreted fungal effector AvrStb6. Although AvrStb6 has been extensively studied as an avirulence determinant, its biological function during host colonization remains unknown. Based on the amphipathic nature of the predicted structure of AvrStb6, we hypothesized the effector to function as a membrane-active antimicrobial protein. However, in vitro growth inhibition assays demonstrated that AvrStb6 does not directly inhibit the growth of wheat-associated bacteria across multiple bacterial genera and experimental conditions. Instead, microbiome analyses of wheat apoplastic fluid revealed shifts in bacterial abundance associated with the presence or absence of AvrStb6 in a susceptible cultivar (without Stb6-induced resistance). This prompted us to further explore other putative microbiome-related functions of AvrStb6. In vitro confrontation assays further showed that deletion of AvrStb6 increased the sensitivity of Z. tritici to antagonistic wheat-associated bacteria, particularly Pseudomonas and Pantoea spp. This phenotype was conserved across independent fungal genetic backgrounds and across virulent and avirulent AvrStb6 variants. Fluorescence-based co-culture assays additionally showed reduced fungal growth and increased bacterial proliferation in the absence of AvrStb6 during interactions with Pseudomonas spp., but not with the control bacterium Escherichia coli. Finally, biochemical assays demonstrated that AvrStb6 associates with the Z. tritici cell wall in vitro, whereas other secreted fungal effectors do not. Collectively, our findings identify a previously uncharacterized role of AvrStb6 in protecting Z. tritici from antagonistic wheat-associated bacteria by associating with the fungal cell wall. More broadly, this work highlights that fungal effectors may contribute to microbial competition and ecological adaptation beyond their established roles in host immune recognition. Author summaryPlant pathogens secrete proteins that help them colonize their hosts. Some of these proteins are recognized by plant immune receptors and trigger disease resistance, but their original biological functions often remain unclear. We investigated the role of AvrStb6, a protein produced by the wheat pathogen that causes Septoria tritici blotch. AvrStb6 is best known because it is recognized by a wheat resistance gene, yet its contribution to fungal growth and survival has remained unknown. We initially tested whether AvrStb6 directly inhibits bacteria that live on wheat leaves, but found no evidence that it acts as an antimicrobial protein. Instead, we discovered that AvrStb6 influences interactions between the pathogen and wheat-associated bacteria. Fungal strains lacking AvrStb6 were more sensitive to several bacterial species that naturally occur in wheat, particularly members of the genera Pseudomonas and Pantoea. We also found that AvrStb6 can associate with the fungal cell wall, suggesting that it helps protect the pathogen during encounters with antagonistic bacteria. Our findings reveal an unexpected role for a fungal effector in microbial competition and show that pathogen proteins traditionally studied in the context of plant immunity can also influence interactions with other microbes. This work highlights the importance of considering the broader microbial community when studying plant-pathogen systems.

pathology↗

bioRxiv2026

Comprehensive online two-dimensional nanoLCxCZE-MS for deep top-down proteomics

Understanding proteoforms, i.e., the various molecular forms in which proteins can exist, is important for deciphering biological processes and diseases. While capillary zone electrophoresis (CZE) proved advantageous for proteoform separation, limited sample loading capabilities restrict its application. Here, we present a novel comprehensive two-dimensional nanoLCxCZE-MS platform for deep top-down proteomics (TDP). The 2D platform is highly automated, enabling robust performance and the possibility to perform proteoform quantitation as demonstrated by isobaric labeling experiments. The high orthogonality of reversed-phase LC and CZE leads to a peak capacity of 2200, leading to an increase in the number of identified proteoforms in a human Caucasian colon adenocarcinoma cell lysate sample by a factor of 3 compared to nanoLC-MS. Furthermore, CZE mobilities enable the attribution of many more proteoforms to a certain proteoform family on the MS1-level. Overall, the flexible platform enables highly efficient separation of intact proteoforms combined with sensitive MS-based TDP workflows, both for untargeted and targeted analysis of complex biological samples. Graphical AbstractWe report a robust and automated comprehensive nanoLCxCZE-MS platform for top-down proteomics. In addition to large volume sample injection and separation by hydrophobicity in the nanoLC, the orthogonal separation by CZE in the second dimension leads to a strong increase in peak capacity and, thus, in the number of identified proteoforms. CZE mobilities also enable the attribution of many more proteoforms to a proteoform family on the MS1-level. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=46 SRC="FIGDIR/small/725123v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@1b39bcforg.highwire.dtl.DTLVardef@ba4f9aorg.highwire.dtl.DTLVardef@1c04147org.highwire.dtl.DTLVardef@fdc953_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Small ORF16 modulates the Mtr complex activity in Methanosarcina mazei Goe1 depending on the molecular hydrogen availability

Until recently, small open reading frame (sORF)-encoded proteins of fewer than 100 amino acids, have attracted increasing attention over the past decade after being overlooked due to limitations in conventional detection methodologies. While numerous previously unannotated sORFs have recently been identified in the mesophilic archaeal model system Methanosarcina mazei, the physiological roles of most of their encoded small proteins remain unknown. We report here the functional characterization of sORF16 encoded small protein MtrR (49 amino acids) and show that it localizes oligomerically at the cytoplasmic membrane. There, it interacts with and influences the activity of tetrahydrosarcinapterin S-methyltransferase (Mtr), a key membrane-bound complex involved in energy metabolism. In vitro interaction and in vivo copurification assays revealed interactions between MtrR and the Mtr-complex, and microscale thermophoresis showed specific interactions with the MtrA subunit. Mutant strains lacking sORF16 exhibited significantly impaired growth in the presence of molecular hydrogen (H2), irrespective of the carbon source. We posit that by modulating the activity of the Mtr-complex, MtrR enables the archaeon adapt to changing environmental H2 conditions.

microbiology↗

bioRxiv2024

Deletion of epithelial HKDC1 decelerates cellular proliferation and impairs mitochondrial function of tumorous epithelial cells thereby protecting from intestinal carcinogenesis in mice

BackgroundA metabolic switch favoring glycolysis over aerobic oxidative phosphorylation, namely the "Warburg effect", represents a hallmark of cancer cells. Hexokinases (HK) catalyze the first step of glycolysis, thereby regulating its rate. Dysregulated HKDC1 (HK domain containing 1) expression has been associated with various cancer types and blocking HKDC1 prevents disease progression for hepatic carcinoma T cell lymphoma and lung adenocarcinoma, but its implication for colorectal cancer (CRC) remained unknown. Here, we functionally investigated the role of HKDC1 for intestinal carcinogenesis. MethodsFirst, we analyzed HKDC1 expression in the intestinal mucosa of healthy controls (HC) and CRC patients and in different tumor tissues using transcriptomic data from publicly available databases. We then generated HKDC1-deficient human and murine colonic epithelial cell lines as well as intestinal organoids and profiled their phenotypic functions. Next, we screened for proteins interacting with HKDC1 by immunoprecipitation. Finally, we generated tumor-bearing ApcMin/+ mice with a conditional deletion of HKDC1 in intestinal epithelial cells and also performed a xenograft mouse model to test the role of HKDC1 for intestinal carcinogenesis in vivo. ResultsHKDC1 was found to be overexpressed in tumor compared to normal tissue of CRC patients. In vitro, HKDC1-deficient human Caco-2 and murine CMT-93 cells displayed reduced proliferation, altered susceptibility to cell death induction, and disrupted mitochondrial functions, particularly mitochondrial respiration. These altered cancer hallmarks were then corroborated in HKDC1-deficient normal and tumor-derived ApcMin/+ intestinal organoids. Immunoprecipitation and mass spectometry proteomic analyses revealed interactions of HKDC1 with several mitochondria-related proteins. In vivo, two distinct mouse models demonstrated that epithelial deletion of HKDC1 protected from carcinogenesis. First, ApcMin/+-Hkdc1{Delta}IEC mice showed mildly improved disease phenotypes in the colon accompanied with reduced numbers of Ki67-positive proliferating epithelial cells. Finally, HKDC1-deficient Caco-2 cells completely failed to form any tumor mass in a xenograft model when implanted into immunodeficient mice. ConclusionsWe demonstrate that HKDC1 influences cancer cell proliferation and susceptibility to cell death, potentially through interactions with mitochondrial proteins that regulate membrane permeability, ultimately impacting intestinal carcinogenesis. Collectively, these findings highlight the significance of HKDC1 for CRC pathobiology, presenting it as a promising target for further investigation and potential therapeutic interventions.

cancer biology↗

bioRxiv2024

Combined cellular and proteomics approach suggest differential processing of native and foreign vibrios in the sponge Halichondria panicea

Phagocytosis is a conserved cellular mechanism for food uptake, defense and general animal-microbe interactions in metazoans. How the discrimination and subsequent processing of different microbes in marine invertebrates is facilitated remains largely unknown. Thereto, we combined a recently developed phagocytic assay with proteomics analysis to compare the phagocytic activity of the sponge Halichondria panicea upon encounter with a native (i.e., H. panicea isolate; Hal 281) and a foreign (i.e., Nematostella vectensis isolate; NJ 1) Vibrio. The sponge cell fraction was recovered after Vibrio exposure of 30 and 60 min and used for cellular (fluorescence-activated cell sorting and microscopy) and proteomics analyses. While the number of phagocytic active cells was similar between the isolates (p = 0.19), the distribution of vibrios over cell types differed (p = 0.02) with the tendency for a higher accumulation of foreign vibrios in small phagocytic cells than the native vibrios. Initially, both vibrios elicited a mitochondrial proteomic response (e.g., TBXASI, CASP7), whereas after 60 min differentially abundant proteins were dominated by lysosomal proteins (e.g., AGA, FUCA, SCARB2) in response to the native compared to immune-related proteins (e.g., AQP9, H2A, SAMHDI) in response to the foreign vibrio. H. panicea may discriminate native and foreign vibrios post-cellular internalization into choanocytes, followed by differential cellular and molecular processing: Native vibrios are readily transferred to archeocytes and elicit a digestive response, whereas foreign vibrios further accumulate before being transferred and provoke an immune response. These findings provide a mechanism for immune specificity in sponges. ImportanceMetazoans recognize and discriminate between native (i.e., associated and/or commonly encountered) and foreign microbes. In marine invertebrates, the mechanisms of microbial discrimination and immune specificity are not well understood. Phagocytosis is a conserved cellular process from amoeba to humans that facilitates the ingestion and digestion of microbial cells and likely plays a role in this discrimination. To elucidate the molecular and cellular basis of immune specificity in marine invertebrates, we examined the differential phagocytic processing of native and foreign Vibrio isolates in a marine sponge. Our findings revealed that both vibrios provoke a generalist mitochondrial protein-mediated response, while native vibrios trigger a digestive response, and foreign vibrios elicit an immune response after being internalized into sponge cells. This study uncovers a mechanism for immune specificity in an early-divergent metazoan and provides a valuable model for studying the evolution of immunity and its role in animal-microbe interactions.

cell biology↗

bioRxiv2024

An ancient lysozyme in placozoans

Lysozymes are an essential part of immunity and nutrition in metazoans, degrading bacterial cell walls via the hydrolysis of peptidoglycan. Although various lysozymes have been reported for higher animals, the origin of animal lysozymes remains elusive as they seem to be lacking in all early branching phyla. In this study, we investigated a putative goose-type lysozyme (PLys, glycoside hydrolase family 23, GH23) of the placozoan Trichoplax sp. H2. We show that PLys is highly active and produced in gland cells of the ventral epithelium. PLys contains a protective and non-conserved cysteine-rich domain N-terminal of the conserved GH23 lysozyme domain. A truncation of this N-terminal domain in the maturation process of PLys leads to a drastic increase in enzymatic activity at the cost of stability. As the lysozyme is most active under acidic conditions, we investigated the pH trajectories during extracellular digestion in situ. Using a pH-senstive fluorescence reporter, we show that Trichoplax sp. H2 acidifies its temporary feeding grooves pulsatively during digestive events close to the optimum pH for PLys activity. To elucidate the evolutionary origin of the metazoan GH23 lysozyme family, we applied a structure-based phylogenetics approach to show that the metazoan g-type GH23 lysozymes originated from a horizontal gene transfer event from bacteria to an early pre-bilaterian ancestor. GH23 lysozymes have then been retained and expanded in many phyla, including Porifera, Cnidaria, Placozoa and chordates, acting as first animal lysozyme and a key component in the antibacterial arsenal since early animal evolution.

evolutionary biology↗

bioRxiv2024

Protein aggregation is a consequence of the dormancy-inducing membrane toxin TisB in Escherichia coli

Bacterial dormancy is a valuable strategy to survive stressful conditions. Toxins from chromosomal toxin-antitoxin systems have the potential to halt cell growth, induce dormancy and eventually promote a stress-tolerant persister state. Due to their potential toxicity when overexpressed, sophisticated expression systems are needed when studying toxin genes. Here, we present an optimized plasmid expression system for toxin genes based on an artificial 5 untranslated region. We applied the system to induce expression of the toxin gene tisB from the chromosomal type I toxin- antitoxin system tisB/istR-1 in Escherichia coli. TisB is a small hydrophobic protein that targets the inner membrane, resulting in depolarization and ATP depletion. We analyzed TisB-producing cells by RNA- sequencing and revealed several genes with a role in recovery from TisB-induced dormancy, including the chaperone genes ibpB, spy and cpxP. The importance of chaperone genes suggested that TisB- producing cells are prone to protein aggregation, which was validated by an in vivo fluorescent reporter system. We moved on to show that TisB is an essential factor for protein aggregation upon DNA damage mediated by the fluoroquinolone antibiotic ciprofloxacin in E. coli wild-type cells. The occurrence of protein aggregates correlates with an extended dormancy duration, which underscores their importance for the life cycle of TisB-dependent persister cells. ImportanceProtein aggregates occur in all living cells due to misfolding of proteins. In bacteria, protein aggregation is associated with cellular inactivity, which is related to dormancy and tolerance to stressful conditions, including the exposure to antibiotics. In Escherichia coli, the membrane toxin TisB is an important factor for dormancy and antibiotic tolerance upon DNA damage mediated by the fluoroquinolone antibiotic ciprofloxacin. Here, we show that TisB provokes protein aggregation, which in turn promotes a deeper state of cellular dormancy. Our study suggests that protein aggregation is a consequence of membrane toxins with the potential to affect the duration of dormancy and the outcome of antibiotic therapy.

microbiology↗

bioRxiv2024

Combining systems and synthetic biology for in vivo enzymology

Enzymatic parameters are classically determined in vitro, under conditions that are far from those encountered in cells, casting doubt on their physiological relevance. We developed a generic approach combining tools from synthetic and systems biology to measure enzymatic parameters in vivo. In the context of a synthetic carotenoid pathway in Saccharomyces cerevisiae, we focused on a phytoene synthase and three phytoene desaturases, which are difficult to study in vitro. We designed, built, and analyzed a collection of yeast strains mimicking substantial variations in substrate concentration by strategically manipulating the expression of geranyl-geranyl pyrophosphate (GGPP) synthase. We successfully determined in vivo Michaelis-Menten parameters (KM, Vmax and kcat) for GGPP-converting phytoene synthase from absolute metabolomics, fluxomics and proteomics data, highlighting differences between in vivo and in vitro parameters. Leveraging the versatility of the same set of strains, we then extracted enzymatic parameters for two of the three phytoene desaturases. Our approach demonstrates the feasibility of assessing enzymatic parameters directly in vivo, providing a novel perspective on the kinetic characteristics of enzymes in real cellular conditions.

systems biology↗

bioRxiv2023

FLASHQuant: a fast algorithm for proteoform quantification in top-down proteomics

Liquid chromatography-mass spectrometry (LC-MS) based top-down proteomics (TDP) is an essential method for the analysis of intact proteoforms. The accurate quantification of individual proteoforms is a crucial step in identifying proteome-wide alterations in different biological conditions. Label-free quantification (LFQ) is the most common method for proteoform quantification as it requires no additional costly labeling. In TDP, due to frequent co-elution and complex signal structures, overlapping signals deriving from multiple proteoforms complicate accurate quantification. Here, we introduce FLASHQuant for MS1-level LFQ analysis in TDP, which is capable of automatically resolving and quantifying co-eluting proteoforms. FLASHQuant performs highly accurate and reproducible quantification in short runtimes of just a few minutes per LC-MS run. To validate the proteoforms reported by FLASHQuant, we evaluated them with identified proteoforms confirmed by tandem mass spectrometry, which showed high match rates. FLASHQuant is publicly available as platform-independent open-source software at https://openms.org/flashquant/.

bioinformatics↗

bioRxiv2023

The apoplastic space of two wheat genotypes provide highly different environment for pathogen colonization: Insights from proteome and microbiome profiling

The intercellular space comprising the plant apoplast harbors a diverse range of microorganisms. The apoplastic interface represents the main compartment for interactions between proteins produced and secreted by the plant and the microbial endophytes. The outcomes of these interactions can play a role in plant cell wall metabolism, stress tolerance, and plant-pathogen resistance. So far the underlying factors that determine microbiota composition in the apoplast are not fully understood. However, it is considered that cell wall composition, nutrient availability, and the plant immune system are main determinants of microbiota composition. The plant immune system is considered to play a crucial role in modulating microbiota composition through the recognition of specific microbe-associated molecular patterns and the activation of defense responses. Hereby the plant may restrict non-beneficial microbial members and facilitate the propagation of beneficial ones. In this study, we investigated changes in the apoplastic environment during pathogen invasion using wheat as a model system. Infection of wheat with Zymoseptoria tritici, a fungal pathogen, resulted in notable alterations in the apoplast composition, reduced microbial diversity, and the accumulation of antimicrobial defense metabolites. Intriguingly, certain core microbial members persisted even in the presence of pathogen-induced immune responses, indicating their ability to evade or tolerate host immune defenses. To further explore these dynamics, we developed a protocol for extracting apoplastic fluids from wheat leaves and conducted proteome analyses to characterize the dynamic environment of the wheat leaves. Our findings uncovered a highly variable apoplastic environment that selects for microbes with specific adaptations. Notably, a core microbial community enriched in the resistant wheat cultivar exhibited antagonistic activity against Z. tritici, suggesting a potential role in conferring pathogen defense. This study advances our understanding of the dynamic interactions and adaptations of the wheat apoplastic microbiota during pathogen invasion, emphasizing the pivotal role of microbial interactions in pathogen defenses.

microbiology↗

bioRxiv2023

A mitochondrial sirtuin shapes the intestinal microbiota by controlling lysozyme expression

Sirtuins act as cellular sensors in the gut that control a substantial change in gut properties in response to environmental changes. Here we show that the only mitochondrial sirtuin of Drosophila, dSirt4, is strongly up-regulated by a protein-reduced diet. Flies with a dSirt4 defect show strong changes in the protein pattern and physiological properties of their intestine. One of the most notable effects was the strong induction of lysozyme gene expression in the intestine, which also translates into enhanced lysozyme activity. This effect was cell autonomous, as it was also observed in flies with dsirt4 was exclusively silenced in enterocytes of the intestine. Although this strongly increased lysozyme expression, it did not reduce total bacterial load in the intestine, but rather changed the composition of the microbiota by reducing the number of gram-positive bacteria. This effect on microbiota composition can be attributed to the dSirt4-dependent lysozyme expression, as it was absent in a lysozyme-deficient background. dSirt4 deficiency in enterocytes reduced lifespan of flies, which was also observed in those flies experiencing ectopic lysozyme overexpression in enterocytes. This implies that strong lysozyme expression leads to a dysbiotic state associated with reduced lifespan.

cell biology↗

bioRxiv2023

The C. elegans proteome response to two protective Pseudomonas symbionts

The C. elegans natural microbiota isolates Pseudomonas lurida MYb11 and Pseudomonas fluorescens MYb115 protect the host against pathogens through distinct mechanisms. While P. lurida produces an antimicrobial compound and directly inhibits pathogen growth, P. fluorescens MYb115 protects the host without affecting pathogen growth. It is unknown how these two protective microbes affect host biological processes. We used a proteomics approach to elucidate the C. elegans response to MYb11 and MYb115. We found that both Pseudomonas isolates increase vitellogenin protein production in young adults, which confirms previous findings on the effect of microbiota on C. elegans reproductive timing. Moreover, the C. elegans responses to MYb11 and MYb115 exhibit common signatures with the response to other vitamin B12-producing bacteria, emphasizing the importance of vitamin B12 in C. elegans-microbe metabolic interactions. We further analyzed signatures in the C. elegans response specific to MYb11 or MYb115. We provide evidence for distinct modification in lipid metabolism by both mutualistic microbes. We could identify activation of host pathogen defense responses as MYb11-specific proteome signature and provide evidence that the intermediate filament protein IFB-2 is required for MYb115-mediated protection. These results indicate that MYb11 not only produces an antimicrobial compound, but also activates host antimicrobial defenses, which together might increase resistance to infection. In contrast, MYb115 affects host processes such as lipid metabolism and cytoskeleton dynamics, which might increase host tolerance to infection. Overall, this study pinpoints proteins of interest that form the basis for additional exploration into the mechanisms underlying C. elegans microbiota-mediated protection from pathogen infection and other microbiota-mediated traits.

molecular biology↗

bioRxiv2023

Bacterial c-di-GMP plays a key role in the evolution of host-association

Most microbes evolve faster than their hosts and should therefore drive evolution of host-microbe interactions1-3. However, relatively little is known about the characteristics that define the adaptive path of microbes to host-association. In this study we have identified microbial traits that mediate adaptation to hosts by experimentally evolving the bacterium Pseudomonas lurida with the nematode Caenorhabditis elegans. We repeatedly observed the evolution of beneficial host-specialist bacteria with improved persistence in the nematode, achieved by mutations that uniformly upregulate the universal second messenger c-di-GMP. We subsequently upregulated c-di-GMP in different Pseudomonas species, consistently causing increased host-association. Comparison of Pseudomonad genomes from various environments revealed that c-di-GMP underlies adaptation to a variety of hosts, from plants to humans, suggesting that it is fundamental for establishing host-association.

evolutionary biology↗

bioRxiv2023

Closely related Bacteroides of the murine intestinal microbiota affect each other's growth positively or negatively

The mammalian intestine is a unique ecosystem for thousands of bacterial species and strains. How naturally coexisting bacteria of the microbiota interact with each other is not yet fully understood. Here, we isolated formerly coexisting, closely related strains of the genus Bacteroides from the intestines of healthy, wild-derived mice. The effect of one strain on another strains growth was tested in 169 pairs in vitro. We find a vast diversity of growth promoting and growth inhibiting activities. A strong positive effect was observed between two strains with differing metabolisms. Growth inhibition among a subset of strains was associated with the known bacterial toxin bacteroidetocin B. Across all strains, we observed growth promotion more often than growth inhibition. The effects were independent of two strains belonging to the same or different species. In some cases, one species differed in its effect on another according to host origin. These findings on obligate host-associated bacteria demonstrate that closely related and naturally coexisting strains have the potential to affect each others growth positively or negatively. These results have implications for our basic understanding of host-associated microbes and the design of synthetic microbial communities.

microbiology↗

bioRxiv2022

Predicting Peptide HLA-II Presentation Using Immunopeptidomics, Transcriptomics and Deep Multimodal Learning

The human leukocyte antigen (HLA) class II proteins present peptides to CD4+ T cells through an interaction with T cell receptors (TCRs). Thus, HLA proteins are key players in shaping immunogenicity and immunodominance. Nevertheless, factors governing peptide presentation by HLA-II proteins are still poorly understood. To address this problem, we profiled the blood transcriptome and immunopeptidome of 20 healthy individuals and integrated the profiles with publicly available immunopeptidomics datasets. In depth multi-omics analysis identified expression levels and subcellular locations as import sequence-independent features governing presentation. Levering this knowledge, we developed the Peptide Immune Annotator Multimodal (PIA-M) tool, as a novel pan multimodal transformer-based framework that utilises sequence-dependent along with sequence-independent features to model presentation by HLA-II proteins. PIA-M illustrated a consistently superior performance relative to existing tools across two independent test datasets (area under the curve: 0.93 vs. 0.84 and 0.95 vs. 0.86), respectively. Besides achieving a higher predictive accuracy, PIA-M with its Rust-based pre-processing engine, had significantly shorter runtimes. PIA-M is freely available with a permissive licence as a standalone pipeline and as a webserver (https://hybridcomputing.ikmb.uni-kiel.de/pia). In conclusion, PIA-M enables a new state-of-the-art accuracy in predicting peptide presentation by HLA-II proteins in vivo.

bioinformatics↗

bioRxiv2021

Calpains are required for efficient microtubule detyrosination

Detyrosination is a major post-translational modification of microtubules (MT), which has significant impact on MT function in cell division, differentiation, growth, migration, polarity, and intracellular trafficking. Detyrosination of -tubulin occurs via the recently identified complex of vasohibin 1/2 (vash1/2) and small vasohibin binding protein (SVBP). However, there is still remaining detyrosinating activity in the absence of vash1/2/SVBP, and little is known about the regulation of detyrosination. Using cellular and cell-free assays we showed that the calcium-dependent proteases calpains 1 and 2 regulate MT detyrosination. We identified new calpain cleavage sites in the N-terminal disordered region of vash1 using in vitro proteolysis followed by mass spectrometry. However, this cleavage did not affect the detyrosination activity of vasohibin. In conclusion, the regulation of MT detyrosination by calpains occurs via another yet unknown tubulin carboxypeptidase. Importantly, calpains calcium dependency could allow a fine regulation of MT detyrosination. Thus, identifying the calpain-regulated pathway of MT detyrosination can be of major importance for several basic and clinical research and should be focused on in future studies. Summary StatementThe conventional calpains 1 and 2 play an important role in the regulation of microtubule detyrosination in a vasohibin independent way. Thus, they possibly control another still unknown tubulin carboxypeptidase.

cell biology↗

bioRxiv2020

Small protein 26 interacts and enhances glutamine synthetase activity in Methanosarcina mazei

Small ORFs (sORF) encoded small proteins have been overlooked for a long time due to challenges in prediction and distinguishing between coding and non-coding predicted sORFs and in their biochemical detection and characterization. We report on the first biochemical and functional characterization of a small protein (sP26) in the archaeal model organism Methanosarcina mazei, comprising 23 amino acids. The corresponding encoding leaderless mRNA (spRNA26) is highly conserved within numerous Methanosarcina strains on the amino acid as well as on nucleotide level strongly arguing for a cellular function of the small protein. spRNA26 is significantly enhanced under nitrogen limitation, but also under oxygen and salt stress conditions. His-tagged sP26 was heterologously expressed and purified by fractionated ammonium sulfate precipitation, affinity chromatography and size exclusion centrifugation. Using independent biochemical approaches (pull-down by affinity chromatography followed by MS analysis, revers pull-down, microscale thermophoresis and size exclusion chromatography) we observed that sP26 interacts and forms complexes with M. mazei glutamine synthetase (GlnA1) with high affinity (app. KD = 45 +/- 14 {micro}M). Upon interaction with sP26, GlnA1 activity was significantly stimulated independently and in addition to the known activation by the metabolite 2-oxoglutarate. Besides strong interaction of sP26 with the PII-like protein GlnK1 was demonstrated (KD= 1.4 {micro}M +/- 0.9 {micro}M). On the basis of these findings, we hypothesize that in addition to 2-oxoglutarate, sP26 activates GlnA1 activity under nitrogen limitation most likely by stabilizing the dodecameric structure of GlnA1.

biochemistry↗

bioRxiv2019

The inducible response of the nematode Caenorhabditis elegans to members of its natural microbiome across development and adult life

The biology of all organisms is influenced by the associated community of microorganisms. In spite of its importance, it is usually not well understood how exactly this microbiome affects host functions and what are the underlying molecular processes. To rectify this knowledge gap, we took advantage of the nematode C. elegans as a tractable, experimental model system and assessed the inducible transcriptome response after colonization with members of its native microbiome. For this study, we focused on two isolates of the genus Ochrobactrum. These bacteria are known to be abundant in the nematodes microbiome and are capable of colonizing and persisting in the nematode gut, even under stressful conditions. The transcriptome response was assessed across development and three time points of adult life, using general and C. elegans-specific enrichment analyses to identify affected functions. Our assessment revealed an influence of the microbiome members on the nematodes dietary response, development, fertility, immunity, and energy metabolism. This response is mainly regulated by a GATA transcription factor, most likely ELT-2, as indicated by the enrichment of (i) the GATA motif in the promoter regions of inducible genes and (ii) of ELT-2 targets among the differentially expressed genes. We compared our transcriptome results with a corresponding previously characterized proteome data set, highlighting a significant overlap in the differentially expressed genes and the affected functions. Our analysis further identified a core set of 86 genes that consistently responded to the microbiome members across development and adult life, including several C-type lectin-like genes and genes known to be involved in energy metabolism or fertility. We additionally assessed the consequences of induced gene expression with the help of metabolic network model analysis, using a previously established metabolic network for C. elegans. This analysis complemented the enrichment analyses by revealing an influence of the Ochrobactrum isolates on C. elegans energy metabolism and furthermore metabolism of specific amino acids, fatty acids, and also folate biosynthesis. Our findings highlight the multifaceted impact of naturally colonizing microbiome isolates on C. elegans life history and thereby provide a framework for further analysis of microbiome-mediated host functions.

evolutionary biology↗